[CANCER RESEARCH 58, 4113-4116. September 15. 1998]

Parathyroid Hormone-related Protein in Patients with Primary Breast Cancerand Eucalcemia1

Elisabet Bucht,2 Haiqin Rong, Ylva Pernow, Ann-Christin Sandberg Nordqvist, Elina Eriksson, Wayne Rankin,

Eva von Schoultz, William J. Burtis, Barbro Granberg, Ursula G. Falkmer, Douglas W. Burton, andLeonard J. DeftosDepartment of Molecular Medicine, Endocrine and Diabetes Unii [E. B., H. R., Y. P., B. G.] and Neurogenetic Unit IA-C. S. N.]. Department of Oncology-Pathology /£.£..E. v. S.I. Karolinska Hospital and Institute, 5-777 76 Stockholm, Sweden; St. Vincent's Institute of Medical Research, 3065 Melbourne, Australia ¡W.R.j; Department of

Medicine, Division of Endocrinology and Metabolism, Veterans Affairs Medical Center, West Haven, Connecticut 06516 [W. J. B.J; Department of Oncology, University Hospitalof Trondheim, N-7006 Trondheim, Norway [V. G. F.]; and University of California, San Diego and the San Diego Veterans Affairs Medical Center, La Jolla, California 92161

[D. W. B., L J. D.I

ABSTRACT

Parathyroid hormone-related protein (PTHrP) is a causative factor of

humoral hypercalcemia in breast cancer and other malignancies. Westudied circulating PTHrP levels with three different immunoassays directed against different parts of the PTHrP molecule in 48 patients withbreast cancer and eucalcemia. The methods used were: (a) a RIA withantibodies directed toward the midregion (63-78); (ft) an ¡mmunofluoro-metric assay with two antibodies against 1-34 and 38-67; and (c) animmunoradiometric assay with antibodies against 1-40 and 1—72.Al

though most patients had PTHrP levels indistinguishable from normalwhen measured by all three methods, four patients had increased serumlevels in the IFMA. PTHrP was detected by immunohistochemistry intumors from nearly all patients. One patient with elevated PTHrP inplasma measured by IFMA showed intense staining of tumor by immunohistochemistry; the tumor was li¡stologicali y graded as III (severe) andwas the largest of all tumors in this patient group. The IFMA can identifyincreased serum PTHrP in some patients with breast cancer who are nothypercalcémie. This assay may be especially useful in screening patientsfor this tumor during a relative early phase of the disease.

INTRODUCTION

PTHrP3 is widely expressed in malignant tumors and seems to be

the main factor causing HHM (1). PTHrP was first isolated in 1987from a breast cancer in a hypercalcémiepatient (2). Since then, thebiological and clinical importance of PTHrP in breast cancer hasbecome well established (3, 4). PTHrP production by breast cancers isvery common, occurring in 50-60% of cases (5), with even higherincidence when the patient is hypercalcémie(6-8). Breast tumors that

produce PTHrP are more likely to metastasize, and breast cancers thatmetastasize to bone are even more likely to produce PTHrP (9). Breastcancers commonly express the PTHrP receptor, as do breast cancercell lines and primary cultures (5, 10, 11). PTHrP and its peptides aresecreted into blood by such breast cancers, and they can serve astumor and serum markers for this malignancy (6, 8). PTHrP is alsopresent in nonmalignant breast tissue studied by immunohistochemistry (12), produced in the lactating mammary gland (13), and presentin milk in high concentrations ( 14). Of the three isoforms generated bymRNA splicing, PTHrP-141 seems to be the dominant gene product.

In addition to mRNA splicing, processing of PTHrP into peptides is an

Received 4/20/98; accepted 7/17/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with¡8U.S.C. Section 1734 solely to indicate this fact.

1This study was supported by Grants 3129 EB and 3921 A-C SN from the Swedish

Cancer Society, Grant 5992 EB from the Swedish Medical Research Council, the Fundsof the Karolinska Institute, and by the NIH and the Department of Veterans Affairs (toL. J. D.).

2 To whom requests for reprints should be addressed, at Department of Molecular

Medicine, Endocrine and Diabetes Unit, Karolinska Hospital L 1:02, S-171 76 Stockholm,Sweden. Fax: 46 8 30 34 58; E-mail: bucht@enk.ks.se.

3 The abbreviations used are: PTHrP, parathyroid hormone-related protein; IFMA,

immunofluorometric assay; IRMA, immunoradiometric assay; HHM. humoral hypercalcemia of malignancy.

important regulatory mechanism (15, 16). Distinct biological properties have been attributed to the different PTHrP peptides, and specificreceptors and effects have been identified (17-19). Intact PTHrP is

cleaved by specific enzymes into smaller fragments. The NH2 terminus has homology with PTH and acts via PTH receptors. PTHrP1-141 contains several amino acid residues that allow it to be cleavedinto many different fragments. For example, PTHrP 1-34 and 1-36mediate the growth-regulating and hypercalcémieeffects of the molecule, PTHrP 35-94 promotes placenta! calcium transfer, and peptidesincluded in the PTHrP 109-141 inhibit osteoclast function (19).

Secretory and circulating forms of PTHrP are being characterized. Alarge NH2 terminal species detected by a PTHrP (1-74) immunora

diometric assay has been demonstrated in patients with HHM (20). Arenally cleared COOH-terminal species, detected by a PTHrP (109-

138) RIA, has been found in elevated concentrations in patients withrenal insufficiency (21). A midregion peptide that begins at aminoacid 38 of the parent protein is secreted into conditioned medium bythree different cell lines (22), and a similar or identical peptide hasalso been demonstrated in patients with HHM (23).

Most studies of PTHrP and breast cancer have focused on hypercalcemia, and there have been few studies of PTHrP in patients withbreast cancer who are not hypercalcémie.The aim of this study wasto investigate PTHrP expression and secretion in breast cancer patients who are not hypercalcémie.

MATERIALS AND METHODS

Patients with primary breast cancer (n —48), diagnosed by means ofpreoperative fine-needle aspiration biopsies, were referred to the Department

of Oncology, Karolinska Hospital. The patients were normocalcemic at thetime of surgery. The following prognostic factors were recorded; tumor size(mm), histological type (according to WHO. Histological Typing of BreastTumors, Ed. 2, Geneva, in Neoplasma, 30 (1): 113-120, 1982) and grade (24),

pathological axillary lymph node status, and estrogen and progesterone receptor levels. Receptor levels >0.1 fmol//j.g DNA were considered positive. Thestudy was approved by the Committee of Ethics of the Karolinska Hospital andperformed in accordance with the Declaration of Helsinki. All patients andsubjects had normal renal function as assessed by creatinine measurements.

Blood and Tumor Processing. Preoperatively, blood samples were collected for analysis of PTHrP in tubes containing protease inhibitors (PTHrPCocktail Tubes; Nichols Institute), from all patients (ages 58 ±8 years; range,29-79 years) and from 19 age-matched (61 ±16 years; range, 30-77 years)healthy women. After centrifugation, the plasma was frozen at -80°C in

aliquots until immunoassays were performed. Tumors from the patients werefixed in 10% formalin and embedded in paraffin; 4-^tm sections were sec

tioned for immunohistochemistry.Chemical Analysis. Serum calcium, albumin, phosphate, and creatinine

were measured by routine laboratory methods.Antisera. The antisera used in the RIA was a mixture of two polyclonal

rabbit antisera raised against the midmolecular fragment of PTHrP, amino acidresidues 63-77 (23). In the two-site IFMA, a polyclonal rabbit antiserum 1926against PTHrP 38-67 and another polyclonal sheep antiserum against PTHrP

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PTHRP IN BREAST CANCER

1-34 were used. Both antisera were affinity purified by cyanogen bromide-

activated Sepharose 4B (Pharmacia) to which their respective PTHrP peptideshad been coupled (25. 26). For immunohistochemistry, a mouse monoclonalantibody 8B12 against PTHrP 1-34 was used (27).

RIA. A synthetic midmolecular fragment of PTHrP 63-77 was used forantibody production in rabbits (14) as reference standard and for radioiodina-tion. The samples were extracted by Sep-Pak C18 cartridges (Waters. Milford,

MA) as described previously together with the RIA (28). The detection limitof the assay, based on 2 SD below maximal binding, was 8.8 pmol/1. Thetheoretical detection limit after extraction was 0.44 pmol/1. Intraassay variationwas <9%, and interassay was <15% at all concentrations.

Two-Site IFMA. This method was described and evaluated recently (26).In brief, antiserum 1926 against PTHrP 38-67 was biotinylated using a biotin

labeling kit (Boehringer Mannheim, Biochemica, Germany) according to themanufacturer's instruction. This antibody was added to streptavidin-coated

microtitration strips (Wallac Oy) and used for capturing PTHrP. RecombinantPTHrP 1-84 was used as standard. The signaling antibody was directed againstPTHrP 1-34 and labeled with europium using a dissociation enhancement

lanthanide fluoroimmunoassay Eu labeling kit (Wallac Oy). The labeling yieldwas 8 mol Eu3+/mol IgG with a specific activity of 2.4 X IO6 cps/ng Eu-IgG.

The fluorescence was measured in a dissociation enhancement lanthanidefluoroimmunoassay 1234 plate fluorometer (Wallac Oy).

The detection limit of the assay, based on the binding of the blank plus 2SD, was 0.3 pmol/1. The intraassay coefficient of variation was 9.6% at 0.31,7.5% at 0.63, and 4.5% at 1.25 pmol/1 (n = 6). The interassay coefficient ofvariation was 14.3% at 2.5, 6.4% at 10.0, and 11.4% at 20 pmol/1 (n = 6).There was no cross-reaction with 100 pmol/1 of the PTHrP fragments 1-36,58-77, or PTH 1-84.

Two-Site IRMA. A commercial kit (Nichols Institute) for PTHrP was also

used (29). Two different polyclonal antibodies to synthetic human PTHrP, Ab(1) and Ab (2), were purified by affinity chromatography. One antibody wasproduced against synthetic amino acid residues 60-72 and was coupled tobiotin. The other antibody recognized the NH2-terminal 1-40 and was radio-

labeled for detection. The detection limit of the assay was 0.3 pmol/1. Therewas no cross-reaction with PTH fragments 1-34,53-84,44-68, or PTH 1-84.

Immunohistochemistry. Immunohistochemistry was performed on tumorsamples from 42 patients. The tumor specimen were formalin fixed andparaffin embedded. Following deparaffmization and rehydration, the sectionswere microwave irradiated before incubation with PTHrP antibodies 8B12 (10/xg/ml) at 4°Covernight in a humidified environment to enhance antigen

retrieval. Negative controls consisted of parallel section incubated with equivalent concentrations of preimmune rabbit IgG. The immunostaining was completed with the standard vector ABC kit method, using diaminobenzidine as achromogen. To confirm the specificity of the immunostaining reaction, selected sections were incubated with PTHrP antibody that had been preadsorbedwith 0.5 mg/ml of PTHrP 1-36 (Peninsula Laboratory, Inc., Belmont, CA)

overnight. The PTHrP expressing lung cancer cell line (BEN; Ref. 30) wasused as a positive control. A pellet of cultured cells were fixed in formalin,embedded in paraffin, and sectioned. These sections were included in allstaining batches. Immunohistochemical staining was evaluated by assessingthe staining intensity as follows: 0, no immunoperoxidase staining in tumorcells; +, weak staining; ++, moderate staining; and + + +, strong staining.

RESULTS

PTHrP in Plasma

Two-Site IFMA. Five of 47 patients had measurable levels ofplasma PTHrP (range, 0.3-0.8 pmol/1). One of 16 healthy, age-related

females had measurable PTHrP (0.4 pmol/1; Fig. 1).Midregion RIA. The RIA requires nearly 10 ml of blood and

could be practically performed on only 33 patients. The apparentplasma concentrations of PTHrP were indistinguisable between thebreast cancer and control groups.

IRMA. PTHrP was determined in 42 patients and 18 healthyfemales. The reference range reported by Quest Diagnostics was <1.3pmol/1. As for the RIA, the apparent concentrations of PTHrP in

1.00 -,

0.75 -

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0.50 -

0.25 -

0.00 J

^3

4

•••••••••o•»•»•*••«•

Control

••*»••••••

Patients

IFMA (PTHrP1-67)

Fig. 1. Plasma PTHrP levels expressed as pmol/1 are demonstrated with a two-site

assay (IFMA) in 16 healthy controls and in 47 patients with primary mammary cancer. O,patients in Table 1. , the detection limit of the assay. Four to five patients have levelsof PTHrP above normal.

patients with breast cancer could not be distinguished from normal(data not shown).

Immunohistochemical Staining for PTHrP

Positive staining of PTHrP with the antibody 8B12 (PTHrP 1-34)

was detected in 41 of 42 patients. Cytoplasmic, vesicular, and nuclearstaining was observed. Positive staining was also observed in thenormal tissue surrounding the tumor.The intensity of staining wasweak (+) in 11 tumors (27%), moderate (++) in 22 tumors (54%),and strong (+ + +) in 8 tumors (20%). One of the tumors with themost intense staining is demonstrated in Fig. 2, and it came frompatient 1 in Fig. 1.

Controls. The positive control, the BEN cell line (30), consistentlyshowed strong PTHrP staining. Preadsorption of the antibodiesagainst PTHrP 1-36 completely abolished the staining. Positive stain

ing was not observed when the primary antibody (8B12) was omitted.

Relationship between Plasma PTHrP Levels, PTHrP Staining,and Clinicopathological Factors

When measured by RIA, one patient (Fig. 1, no. 4) had slightlyelevated plasma PTHrP levels but nonelevated concentrations measured by the two other immunoassays; the staining in this patientstumor by immunohistochemistry was ++, and the histological gradeof the tumor was II. By measurement with the IFMA, only one healthysubject had measurable levels (0.4 pmol/1), and five patients hadlevels above the detection limit, 0.3 pmol/1 (Fig. 1). The tumor fromthe patient with the highest plasma PTHrP (Fig. 1, no. 1) was stronglystained by immunohistochemistry with PTHrP antibodies toward the

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PTHRP IN BREAST CANCER

Fig. 2. Photomicrograph shows PTHrP immunoreactive cells ina section from the tumor of patient no. 1 in Table 1 and Fig. 1. Thetumor in this case displayed robust amounts of PTHrP peptidedetected by antibody 8B 12. x, 200; bar, 5 pm.

Table 1 Demonstration of the data on the four patients with plasma PTHrP levelsabove ¡hedetection limit when measured bv the IFMA

PatientPTHrP

measuredbyIFMA(pmol/1)IRMA(pmol/I)RIA

(pmol/1)ImmunohistochemicalstainingHistologicalgradeTumortypeAxillary

lymph nodestatusEstrogenreceptorsProgestronereceptorsSerum

calcium (nM)10.80.52.0+

++II1++DuctalPositiveNegativeNegative2.3620.7<0.31.5+

+—LobularPositivePositivePositive2.1530.5<0.3—++11DuctalNegativePositivePositive-40.40.311.5++IIDuctalPositivePositivePositive2.25

NH2 terminus. The tumor was ductal, histologically graded as III+ +in severity, and had the largest size of all tumors in the study. Thetumor was negative for both estrogen and progesterone receptors. Theimmunological and clinical data on patients with plasma PTHrP abovethe detection limit when measured by IFMA are demonstrated inTable 1. Measurements of a longer fragment by IRMA demonstratedindistinguisable levels in the control group and in the patients, andthere was no correlation between PTHrP and any of the clinicopath-

ological factors that were performed.There was no correlation between the grade of immunohistochem-

ical PTHrP staining and clinicopathological factors (Table 2). Allpatients were normocalcemic, and there was no correlation betweenand among serum calcium and PTHrP measured by any of the threemethods.

DISCUSSION

Increased production of PTHrP in breast cancers is associated withthe hypercalcemia that commonly accompanies this tumor. However,there are few studies that focus on circulating PTHrP in patients withbreast cancer who are not hypercalcémie.Our studies demonstratedthat in 47 such patients, we were able to identify four patients withelevated serum concentrations of PTHrP and one patient with levels atthe upper limit of normal (Fig. 1) with an IFMA based on PTHrP1-67. By contrast, two other assays failed to distinguish betweennormal subjects and breast cancer, an IRMA based on PTHrP 1-74and a RIA based on 63-78. The most likely explanation for this

finding could be that the molecular forms of PTHrP that were secreted

by the breast cancers correspond more closely to the PTHrP 1-67

form detected by IFMA, and the IFMA may be inherently moresensitive in detecting the molecular forms of circulating PTHrP.

The absence of hypercalcemia in our subjects may represent anearly phase of the disease, because our observation time was only 2years. The presence of a robust antihypercalcemic homeostasis inthese patients could also attenuate hypercalcemia. This homeostasiscould be mediated by a variety of factors such as decreased PTHsecretion, decreased 1,25-(OH)2 vitamin D production, and/or in

creased calcitonin secretion (31). Although we were unable to makethese measurements in our patients, these calcémiehormones shouldbe measured in future studies of PTHrP in breast cancer.

Our studies demonstrate that measurements of plasma levels ofPTHrP in some patients with breast cancer can serve as a plasma andtumor marker for the cancer in the absence of hypercalcemia andpresumably in a relative early phase of the disease, before hypercalcemia develops. In such patients, certain PTHrP assays might beuseful for screening to identify them early and to monitor therapy.Although most of the patients we studied had normal levels when

Table 2 Distribution of clinical and pathological variables according Io PTHrPdetected b\ immunohistochemistn'

Clinical and pathologicalvariablesHistologyLobularLobular/tubularLobular/ductalDuctalOthersHistological

gradeIninAxillary

nodestatusNegativePositiveEstrogen

receptorNegativePositiveProgesterone

receptorNegativePositiveTumor

size(mm)Mean±SDTotal

numberImmunohistochemistry

stainingdensityNo.41129651119251610281523410

+1171

11

1341

6541

51

546

16 ±7110+

+3163361113841651520

±1122+

++16112453264425

±24S

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measured by three assays, the IFMA identified four and perhaps fivepatients with elevated levels (Fig. 1). This observation could indicatethat there is a circulating form of PTHrP extending from the NH2terminus to amino acid residues 58 or 66, where basic amino acidresidues are situated. Edwards et al. (32) found no measurable plasmalevels of PTHrP 1-86 in 46 unselected patients with breast cancer

prior to surgery, although 32 of them had tumors with positivestaining of PTHrP. Assay sensitivity notwithstanding, this suggeststhat assays measuring fragments longer than 66 amino acid residuesdo not detect clinically significant circulating PTHrP fragments. Thereare possible cleavage sites at amino acid residue 66 (arginine) and 58(arginine), and our novel assay (IFMA) is likely detecting a fragmentextending from the NH2 terminus to one of these amino acid residues.

Immunostaining for PTHrP was observed in nearly all tumors to alesser extent and in the normal breast tissue surrounding the tumors.This is in accordance with studies by Liapis et al. (12), who foundimmunoreactive protein within the cytoplasm of lobular and ductalepithelial cells in all normal and fibrocystic mammary tissues from 74patients using antibodies against the middle and COOH-terminal

domains of PTHrP and by in situ hybridization of PTHrP mRNA. Thisis not surprising because PTHrP is expressed in most tissues and isregarded as a normal product of mammary epithelial cells (33).Synthesis and secretion of PTHrP has also been demonstrated innormal human mammary epithelial cells in culture (34). In our studies, there was no correlation between plasma levels of PTHrP and theintensity of the immunostaining; even the patient with the highestlevels of plasma PTHrP measured by IFMA had intense staining. Inagreement with other studies (12, 5), we found no correlation betweenPTHrP levels and hormone receptor status. It could be valuable tomeasure a shorter NH2-terminal fragment of PTHrP, because there areseveral cleavage sites in this peptide, and the NH2 terminus 1-36 is an

active secretory form of the molecule.

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1998;58:4113-4116. Cancer Res   Elisabet Bucht, Haiqin Rong, Ylva Pernow, et al.   Breast Cancer and EucalcemiaParathyroid Hormone-related Protein in Patients with Primary

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